Development of safe and efficient gene delivery systems is a critical need for successful gene therapy. Much progress has been made in improving transfection efficiency of cationic lipid/DNA delivery systems in vivo based on lipid structure-activity relationships and lipid/DNA formulation studies. However, several problems remain. Administration of formulations with high lipid/DNA ratios causes some toxicity. Transfected DNA is rapidly degraded limiting the duration of transgene expression. Transgene expression is markedly reduced with repeated administration. We have synthesized a large number of new cationic lipids, several of which appear to be more effective at delivering DNA and less toxic than known compounds in vivo. We have also developed a formulation that allows efficient transfection at much reduced lipid/DNA ratios. Based on these preliminary observations and data in the literature, we hypothesize that: 1) in vivo transfection efficiency and toxicity of lipid/DNA complexes are related to lipid structure and relationships can be defined which provide a rationale for designing lipids and lipid/DNA formulations with optimal efficiency/ toxicity profiles, 2) inhibition of effective transfection by lipid/DNA complexes by serum is a result of interaction of the complexes with specific serum components and defining these interactions will permit design of formulations which are less inhibited by blood components, 3) the magnitude and duration of in vivo expression of plasmid are affected by the host's inflammatory response to the foreign DNA, and this response can be minimized by altering the physical and chemical state of DNA and inhibitors to the response. To test these hypotheses, we propose: 1) to use a new cationic lipid (MMET) as a model to design and test improved lipids for in vivo DNA delivery based on defined structure-activity relationships, 2) to characterize in detail the interaction of serum with lipid/DNA complexes, the tissue distribution of transgene expression and in vivo toxicity profile of new lipid/DNA formulations, 3) in mice, to determine inflammatory responses to lipid/DNA transfection, and relate these response to the magnitude and duration of transgene expression following single and repeated dosing and 4) to determine whether the inflammatory response and the magnitude and duration of transgene expression can be affected favorably by either altering the physical and chemical properties of DNA, or molecular and pharmacological interventions to manage the inflammatory response.